Abstract Stroke is a major medical concern for United States military veterans. Disruption of the blood-brain barrier (BBB) is a catastrophic event in the pathogenesis of ischemic/reperfusion (I/R) brain injury. Our recent studies suggested that structural alterations in brain endothelial cells (EC), including abnormal actin polymerization and the resulting redistribution of junctional proteins, is a novel mechanism responsible for early BBB leakage after I/R (30 min-3h); while matrix metalloproteinase (MMP) 2/9 activation, a predominant mechanism thought to contribute to post-stroke BBB disruption, contribute to the BBB leakage to larger molecules (>40kDa) in a relatively delayed manner. Therefore, restoring EC structure/function may offer an innovative therapeutic strategy for early BBB protection against I/R, while blocking MMP2/9 may provide a relatively delayed protection to BBB. Heat shock protein 27 (HSP27), a member of the small heat shock protein family, confers neuroprotection in several models of CNS diseases, including I/R brain injury. In addition to its well-known protein chaperone and anti-apoptotic functions, HSP27 may act as a potent actin depolymerization factor in certain cell types such as EC, thus potentially inhibiting actin polymerization-mediated BBB disruption. Further, HSP27 may inhibit the activation of NF-kappaB, a central signaling molecule for the production of MMP2/9 and pro-inflammatory mediators. We recently observed that HSP27 is transiently upregulated in brain EC after I/R. However, the precise role of HSP27 in the functional integrity of EC following I/R and the underlying mechanism remain unknown. Using transgenic mice overexpressing HSP27 and the lentiviral gene-transfection approach, we have obtained novel pilot data showing that HSP27 overexpression protects the endothelium from I/R-induced hyperpermeability in vitro and in vivo; that HSP27 inhibited oxygen glucose deprivation-induced actin polymerization and redistribution of junctional proteins in EC; and that intravenous administration of HSP27 containing a cell permeable transduction domain (TAT-HSP27) enabled rapid delivery of the protein into brain micro-vasculatures, reduced BBB damage and inhibited MMP2/9 activity after I/R. This proposal will further explore the BBB protective effect of HSP27 on I/R brain injury and elucidate the underlying mechanisms. The central hypothesis to be tested is that HSP27 protects BBB against I/R injury by stabilizing EC cytoskeletal organization in microvasculature early after I/R and inhibiting the production of MMP2/9 as well as pro-inflammatory proteins. The following specific aims are proposed: Aim 1: To determine whether endothelial targeted overexpression of HSP27 is sufficient to provide early protection on BBB integrity and to confer long term protection against cerebral I/R. Aim 2: Test the hypothesis that HSP27 protects endothelial integrity following I/R by dualistic mechanisms: stabilizing the actin cytoskeleton and inhibiting NFkB-dependent MMP2/9 production and inflammation. Aim 3: Test the hypothesis that post-stroke delivery of TAT-HSP27 to EC protects against BBB damage and improves outcomes in both young adult and aged mice. The proposed study attempts to develop HSP27 into a novel, clinically feasible therapeutic strategy to ameliorate post-stroke BBB damage, brake down the progression of brain damage, and improve long-term neurological functions in stroke victims. The successful completion of this proposed study will help improve the quality of life for veterans suffering from stroke.